Glutathione sepharose 4b resin

The pGEX-4 T-1 harboring recombinant defensin (pGEX-4 T-1-Def) was transformed into the BL21 (DE3) E. coli strain (Elgaied et al. 2017 (link); Elmenofy et al. 2020a (link)). Positive colonies were selected on the basis of their growth on Luria–Bertani (LB) agar plates supplemented with 50 µg/mL ampicillin. The 5 mL LB medium containing ampicillin was inoculated with a single isolated colony and grown overnight at 37 °C. The expression of the GST-defensin fusion was induced by the addition of 0.1 mM IPTG. The bacterial pellet was collected by centrifugation, and the recombinant protein was batch purified with Glutathione Sepharose 4B resin (Sigma, St Louis, USA). In an overhead shaker, the filtered bacterial lysate was incubated with 2 mL of glutathione Sepharose and left overnight at 4 °C. The unbound proteins were washed twice with 10 mL of GST binding buffer, followed by two washes with 10 mL of GST binding buffer containing 1% Triton X-100 to remove nonspecifically bound proteins. The bound recombinant GST-defensin peptide was eluted with 1 mL of elution buffer (50 mM Tris–HCl pH 8.0, 400 mM NaCl, and 10 mM reduced glutathione). The N-terminal GST-tag was cleaved by overnight digestion of thrombin, and then the purity of the recombinant protein was analyzed by Tris-Tricine gel electrophoresis, and its concentration was estimated by Bradford assay.

The cDNA of the maize 14-3-3 isoform GF14-6 (G-BOX FACTOR14-6) was cloned into the pGEX-2T vector, expressed in Escherichia coli as a GST-fused protein [35 (link)]. The GST-14-3-3 was immobilized onto a Glutathione Sepharose 4B resin (Merck KGaA, Darmstadt, Germany), and the 14-3-3 protein eluted by thrombin cleavage as already described [69 (link)]. The overlay assay was performed according to Visconti et al. [70 (link)], with slight modifications. Ten micrograms of two-phase partitioned plasma membrane material was subjected to SDS-PAGE and blotted onto a PVDF membrane. The membrane was blocked with 5% fatty-acid-free milk in buffer H (25 mM Hepes-OH, 75 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 0.04% Tween-20, pH 7.5) and then incubated overnight at 4 °C in buffer H containing 3% fatty-acid-free milk and 50 µg/mL 14-3-3. The membrane was washed extensively with buffer H and incubated with the anti-GF14-6 antibody (1:600) in the same buffer. Immunodecoration was performed as indicated in Section 4.6. The densitometric analysis was performed using ImageJ image-processing software [64 ]. The densitometric values are expressed as a percentage of the maximum integrated densitometric value (the product of the area and mean grey value).